Novel multi-color immunofluorescence technique using primary antibodies raised in the same host species.

Novel multi-color immunofluorescence technique using primary antibodies raised in the same host species.

Alex Kalyuzhny*¹, J.P. Houchins¹, Michael Grahek¹, Jillian Frisch¹, J.M. Schoephoerster¹, Jodi Hagen¹, Joseph Sweet¹, Leopoldo Mendoza² and David Schwartz^{2
1R&D Systems, Inc., Minneapolis, MN 55413 and 2SoluLink, Inc. San Diego, CA92121.}

ABSTRACT

Simultaneous detection of multiple tissue antigens is one of the most frequently used immunohistochemical (IHC) techniques. In order to avoid cross-reactivity of secondary antibodies with unwanted primary antibodies when doing either dual- or triple-labeling immunofluorescence it is necessary to use primary antibodies raised in different host species such as mouse, rabbit and goat antibodies. However, in many cases suitable primary antibodies raised in different species are unavailable. We have developed a novel technique for triple-labeling immunofluorescence IHC that can be used with primary antibodies derived from a single host source. This technique includes modification of one primary antibody with biotin (ChromaLink™ Biotin) and a second primary antibody with DIG (ChromaLink™ Digoxigenin). For IHC staining, cells or tissue sections are incubated first with unconjugated primary antibody against the 1st target protein followed by detection with anti-primary secondary antibody conjugated to NorthernLights™ NL-493 tag (green fluorescence). Subsequently, the same tissue sections are incubated with a mixture of same species biotin-labeled primary antibody (against the 2nd target protein) and DIG-labeled primary antibody (against the 3rd target protein) followed by detection using a mixture of Streptavidin NorthernLights™ NL-557 tag (red fluorescence) and anti-DIG secondary antibody conjugated to a Cy5™ tag (fluorescence in the far red spectral region).

This novel technique provides good spectral separation of colors depicting different antigens of interest while avoiding cross-reactivity between irrelevant primary and secondary antibodies. In addition, this multiplexed IHC technique provides significant convenience to researchers who have at their disposal only primary antibodies raised in the same host species.

INTRODUCTION

Multi-color immunofluorescence histochemistry is one of the most frequently used techniques in neuroscience research. It allows researchers to study temporal and spatial co-localization of different neuronal targets. To avoid cross-reactivity of secondary antibodies with primary antibodies bound to neuronal proteins, researchers have to employ primary antibodies raised in different host species. However, in many cases the most specific primary antibodies available are raised in the same host species precluding investigators from doing multi-color IHC. In order to use primary antibodies raised in the same host species it would be necessary to modify the primary antibodies so they are not binding with unwanted secondary antibodies.

The purpose of this study was to investigate whether unmodified primary antibodies combined with primary antibodies raised in the same host species and modified via either biotinylation or labeling with digoxigenin (DIG) can be used for multi-color fluorescecence IHC.

We have tested our technique using goat, mouse monoclonal, and rabbit primary antibodies and developed a simple and reliable protocol that can be used on cryostat sections of rat and mouse brain.

MATERIALS & METHODS

Animals:

Rats (Sprague Dawley, 200 g) and mice (C57/Bl, 40 g) were deeply anesthetized with sodium pentobarbital and transcardially perfused with PBS followed by Lana’s fixative and 10% sucrose. Brain was cut into 10 micron thick cryostat sections.

R&D Systems’ Primary Antibodies:

Goat (affinity purified):

1. anti-mouse Netrin-4 (Cat #: AF1132)
2. anti-mouse E-Cadherin (Cat #: AF748)
3. anti-mouse Neprilysin (Cat #: AF1126)

Mouse (monoclonal):

1. anti-human CART (Cat #: MAB163)
2. anti-human Calbindin D (Cat #: MAB3320)
3. anti-rat Synaptotagmin (Cat #: MAB4364)

R&D Systems’ Secondary Antibodies:

1. Streptavidin NL-493 (Cat #: NL997)
2. anti-mouse NL-637 (Cat #: NL008)
3. anti-goat NL-637 (Cat #: NL002)
4. anti-rabbit NL-637 (Cat #: NL005)

Other Secondary Antibodies:

anti-DIG Rhodamine Red™ X (Jackson ImmunoResearch; Cat #: 200-292-156)

IHC Protocol:

Tissue sections were first incubated with unconjugated primary antibodies (against the 1st target protein) followed by incubation with corresponding fluorescent secondary antibodies. Tissue sections were then washed with PBS and incubated with a mixture of same species biotin- labeled primary antibody (against the 2nd target protein) and DIG-labeled primary antibody (against the 3rd target protein) followed by detection using a mixture of fluorescent Streptavidin conjugate and anti-DIG secondary antibody.

Incubation with primary antibodies was done overnight at 4°C.

Incubation with secondary antibodies/Streptavidin conjugate was done for 1 hour at room temperature.

Images were collected using an Olympus FluoView® FV1000 confocal microscope.

SoluLink Conjugation Kits:

1. ChromaLink Biotin One-Shot Antibody Labeling Kit (Cat # : B-9007-009K) LINK
2. ChromaLink Digoxigenin One-Shot Antibody Labeling Kit (Cat #: B-9014-009K) LINK